Division of Allergy and Immunology, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45229; and Rothenberg@cchmc.org.

Abstract

Eosinophilia and its cellular activation are hallmark features of asthma, as well as other allergic/Th2 disorders, yet there are few, if any, reliable surface markers of eosinophil activation. We have used a FACS-based genome-wide screening system to identify transcriptional alterations in murine lung eosinophils recruited and activated by pulmonary allergen exposure. Using a relatively stringent screen with false-positive correction, we identified 82 candidate genes that could serve as eosinophil activation markers and/or pathogenic effector markers in asthma. Carbonic anhydrase IV (Car4) was a top dysregulated gene with 36-fold induction in allergen-elicited pulmonary eosinophils, which was validated by quantitative PCR, immunohistochemistry, and flow cytometry. Eosinophil CAR4 expression was kinetically regulated by IL-5, but not IL-13. IL-5 was both necessary and sufficient for induction of eosinophil CAR4. Although CAR4-deficient mice did not have a defect in eosinophil recruitment to the lung, nor a change in eosinophil pH-buffering capacity, allergen-challenged chimeric mice that contained Car4(-/-) hematopoietic cells aberrantly expressed a series of genes enriched in biological processes involved in epithelial differentiation, keratinization, and anion exchange. In conclusion, we have determined that eosinophils express CAR4 following IL-5 or allergen exposure, and that CAR4 is involved in regulating the lung transcriptome associated with allergic airway inflammation; therefore, CAR4 has potential value for diagnosing and monitoring eosinophilic responses.

A, With > 90% purity, Car4+/+ and Car4−/− bone marrow–derived eosinophils (EOS, 2×106) were assessed for their cellular pH buffering capacity in saline without IL-5 following repetitive addition of 5 μL of 0.1 M HCl into 1 mL of cell suspension each time, with cell-free saline solution alone as negative control without cellular buffering. B, The intracellular pH changes in Car4+/+ and Car4−/− eosinophils were graphed before and after the exogenous lactate challenge (20 minutes), demonstrating no difference in buffering capacity which is required for lactate transport into cells. (mean ± SEM)